The present invention relates to genetic engineering in plants using recombinant DNA technology in general and to enzymes involved in the biosynthesis of cyanogenic glycosides and genes encoding these enzymes in particular. The proteins and genes according to the invention can be used to improve the nutritive value or pest resistance of plants.
Cyanogenic glycosides constitute secondary plant metabolites in more than 2000 plant species. In some instances they are the source of HCN which can render a plant toxic if it is taken as food. For example the tubers of the cyanogenic crop cassava (Manihot esculenta) constitute an important staple food in tropical areas. The cyanogenic glycosides present in the tubers may cause cyanide poisoning in humans due to insufficiently processed cassava products. Other plant species whose enzymatic production of HCN accounts for their potential toxicity if taken in excess as food or used as animal feed include white clover (Trifolium repens), sorghum (Sorghum bicolor), linen flax (Linum usitatissimum), triglochinin (Triglochin maritima), lima beans (Phaseolus lunatus), almonds (Amygdalus) and seeds of apricot (Prunus), cherries and apple (Malus). The toxic properties could be reduced by blocking the biosynthesis of cyanogenic glycosides in these plants.
The primary precursors of the naturally occuring cyanogenic glycosides are restricted to the five hydrophobic protein amino acids valine, leucine, isoleucine, phenylalanine and tyrosine and to a single non-protein amino acid, cyclopentenylglycine. These amino acids are converted in a series of reactions to cyanohydrins which are ultimately linked to a sugar residue. Amygdalin for example constitutes the O-xcex2-gentiobioside and prunasin the O-xcex2-glucoside of (R)-mandelonitrile. Another example of cyanogenic glycosides having aromatic aglycones is the epimeric pair of the cyanogenic glycosides dhurrin and taxiphyllin which are to be found in the genus Sorghum and Taxus, respectively. p-Hydroxymandelonitrile for example is converted into dhurrin (xcex2-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile) by a UDPG-glycosyltransferase. Similiar glycosyltransferases are believed to be present in most plants. Vicianin and lucumin are further examples for disaccharide derivatives similiar to amygdalin. Sambunigrin contains (S)-mandelonitrile as its aglycone and is therefore epimeric to prunasin. Examples of cyanogenic glycosides having aliphatic aglycones are linamarin and lotaustralin found in clover, linen flax, cassava and beans. A detailed review on cyanogenic glycosides and their biosynthesis can be found in Conn, Naturwissenschaften 66:28-34, 1979, herein incorporated by reference.
The biosynthetic pathway for the cyanogenic glucoside dhurrin derived from tyrosine has been extensively studied (Halkier et al, xe2x80x98Cyanogenic glucosides: the biosynthetic pathway and the enzyme system involvedxe2x80x99 in: xe2x80x98Cyanide compounds in biologyxe2x80x99, Wiley Chichester (Ciba Foundation Symposium 140), pages 49-66, 1988; Halkier and Moller, Plant Physiol. 90:1552-1559, 1989; Halkier et al, The J. of Biol. Chem. 264:19487-19494, 1989; Halkier and Moller, Plant Physiol. 96:10-17, 1990, Halkier and Moller, The J. of Biol. Chem. 265:21114-21121, 1990; Halkier et al, Proc. Natl. Acad. Sci. USA 88:487-491, 1991; Sibbesen et al, in: xe2x80x98Biochemistry and Biophysics of cytochrome P450. Structure and Function, Biotechnological and Ecological Aspectsxe2x80x99, Archakov, A. I. (ed.), 1991, Koch et al, 8th Int. Conf. on Cytochrome P450, Abstract PII.053; and Sibbesen et al, 8th Int. Conf. on Cytochrome P450, Abstract PII.016). L-Tyrosine is converted to p-hydroxy-mandelonitrile (the precursor of dhurrin), with N-hydroxytyrosine, N,N-dihydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyd oxime, and p-hydroxyphenylacetonitrile being intermediates. Two monooxygenases of the cytochrome P450 type are involved in this pathway. In cassava a similiar pathway involving cytochrome P450 dependent monooxygenases is used for the synthesis of linamarin and lotaustralin from valine and isoleucine, respectively (Koch et al, Archives of Biochemistry and Biophysics, 292:141-150, 1992). The complex pathway from L-tyrosine to p-hydroxy-mandelonitrile in Sorghum bicolor was demonstrated to require two multi-functional cytochrome P450 dependent monooxygenases only. The first enzyme, designated P450TYR, converts tyrosine to p-hydroxyphenylacetaldehyd oxime. The second enzyme, designated P450OX, converts the aldoxime to p-hydroxy-mandelonitrile. In view of the similiarities between the biosynthetic pathways of cyanogenic glucosides in different plants it is generally assumed that said pathways involve two multifunctional P450 dependent monooxygenases, P450I and P450II, which convert the precursor amino acid to the corresponding aldoxime and the aldoxime to the corresponding cyanohydrin, respectively. P450I is a specific enzyme which determines the substrate specificity and, thus, the type of glucoside produced, whereas P450II is expected to be less specific in converting a range of structurally different aldoximes into the corresponding cyanohydrin. Glucosinolates are hydrophilic, non-volatile thioglycosides found within several orders of dicotyledoneous angiosperms (Cronquist, xe2x80x98The Evolution and Classification of Flowering Plants, New York Botanical Garden, Bronx, 1988). The occurance of cyanogenic glucosinolates and glucosides is mutually exclusive. The greatest economic significance of glucosinolates is their presence in all members of the Brassicaceae (order of Capparales), whose many cultivars have for centuries provided mankind with a source of condiments, relishes, salad crops and vegetables as well as fodders and forage crops. More recently, rape (especially Brassica napus and Brassica campestris) has emerged as a major oil seed of commerce. About 100 different glucosinolates are known possessing the same general structure but differing in the nature of the side chain. Glucosinolates are formed from protein amino acids either directly or after a single or multiple chain extension (Underhill et al, Biochem. Soc. Symp. 38:303-326, 1973). N-hydroxy amino acids and aldoximes which have been identified as intermediates in the biosynthesis of cyanogenic glycosides also serve as efficient precursors for the biosynthesis of glucosinolates (Kindl et al, Phytochemistry 7:745-756, 1968; Matsuo et al, Phytochemistry 11:697-701, 1972; Underhill, Eur. J. Biochem. 2:61-63, 1967). Cytochrome P450I involved in cyanogenic glycoside synthesis is thus functionally very similiar to the corresponding biosynthetic enzyme in glucosinolate synthesis, and is therefore expected to be a member of the same family of P450 enzymes. Thus we have isolated a cDNA clone from Sinapis alba encoding a P450 enzyme (SEQ ID NO:17) with 54% identity to P450TYR (CYP79) and catalyzing the first step in the biosynthesis of glucosinolates, that is the formation of the aldoxime from the parent amino acid. This cDNA clone shows approximately 90% identity to an Aribidopsis EST sequence (T42902) which strongly indicates that this cytochrome P450 enzyme is highly conserved in glucosinolate containing species.
The reduction of the complex biosynthetic pathway for cyanohydrins described above to the catalytic activity of only two enzymes, cytochrome P450I and P450II, allows for the manipulation of the biosynthetic pathway of cyanogenic glucosides in plants. By transfection of gene constructs coding for one or both of the monooxygenases a biosynthetic pathway for cyanogenic glucosides can either be modified, reconstituted, or newly established.
The modification or introduction of a biosynthetic pathway for cyanogenic glycosides in plants by methods known in the art is of great interest, since cyanogenic glycosides can be toxic to insects, acarids, and nematodes. Therefore, the modification, introduction or reconstitution of a biosynthetic pathway for cyanogenic glycosides in plants or certain plant tissues will allow to render plants unpalatable for insects, acarids or nematodes and thus help to reduce the damage to the crop by pests. In combination with other insecticidal principles such as Bacillus thuringiensis endotoxins the damage to the crop by pests could be even further reduced.
Alternatively, the sequences of the genes encoding the monooxygenases according to the invention can be used to design DNA plasmids which upon transfection into a plant containing cyanogenic glycosides such as cassava, sorghum or barley, eliminate cyanogenic glycosides normally produced in wildtype plants. This can be achieved by expression of antisense or sense RNA or of ribozymes as described in EP-458367-A1, EP-240208-A2, U.S. Pat. No. 5,231,020, WO89/05852, and WO90/11682 which inhibits the expression of monooxygenases according to the invention. This is of great interest as in spite of numerous efforts it has not been possible through traditional plant breeding to completely remove the cyanogenic glycosides from for example cassava or sorghum. On the other hand it has been shown that elevated amounts of cyanogenic glycosides in the epidermal cells of barley cultivars confer increased sensitivity to attack by the mildew fungus Erysiphe graminis (Pourmohensi, PhD thesis, Gxc3x6ttingen, 1989; Ibenthal et al, Angew. Bot. 67:97-106, 1993). A similiar effect has been observed in the cyanogenic rubber tree Hevea brasiliensis upon attack by the fungus Microcyclus ulei (Lieberei et al, Plant Phys. 90:3-36, 1989) and with flax attacked by Colletotrichum lini (Lxc3xcdtke et al, Biochem. Z. 324:433-442, 1953). In these instances the quantitative resistance of the plants stipulated above and of other plants, where cyanogenic glycosides confer increased sensitivity to attack by microorganisms, can be increased by preventing the production of cyanogenic glycosides in such plants. In barley, the cyanogenic glycosides are located in the epidermal cells. The expression of antisene, sense or ribozyme constructs is therefore preferably but not necessarily driven by an epidermis specific promoter.
The presence of even minor amounts of cyanogenic glycosides in plants may also cause nutritional problems due to generation of unwanted carcinogens as demonstrated in barley. Barley malt for example contains low amounts of the cyanogenic glucoside epiheterodendrin which in the cause of production of grain-based spirits can be converted to ethylcarbamate which is considered to be a carcinogen. Attempts are being made to introduce mandatory maximum allowable concentrations of ethylcarbamate in fermented food, beverages and spirits (Food Chemical News 29:33.35, 1988).
WO 95/16041 describes a DNA molecule coding for a cytochrome P450I monooxygenase, which catalyzes the conversion of an amino acid to the corresponding N-hydroxyamino acid, N,N-dihydroxyamino acid, and the conversion of the N,N-dihydroxyamino acid to the corresponding aldoxime. The parent amino acid is selected from the group consisting of tyrosine, phenylalanine, tryptophan, valine, leucine, isoleucine and cyclopentenylglycine. The DNA molecules either correspond to naturally occuring genes or to functional homologues thereof which are the result of mutation, deletion, truncation, etc. but still encode a cytochrome P450I monooxygenase capable of catalyzing more than one reaction of the biosynthetic pathway of cyanogenic glycosides. The monooxygenases preferably contain a single catalytic center.
Additionally WO 95/16041 describes DNA molecules coding for cytochrome P450II monooxygenases such as P450OX of Sorghum bicolor (L.) Moench. They catalyze the conversion of an aldoxime to a nitrile and the conversion of the nitrile to the corresponding cyanohydrin. The catalysis of the conversion of tyrosine into p-hydroxyphenylacetonitrile by two multifunctional P450 enzymes explains why all intermediates in this conversion except (Z)-p-hydroxyphenylacetaldoxime are channelled. The strategy suggested for the isolation of P450OX is based on that used for the isolation of P450TYR (CYP79, Sibbesen et al, Proc. Natl. Acad Sci. USA 91: 9740-9744, 1994) from sorghum. In this approach a DEAE Sepharose ion exchange column serves to bind P450 enzymes whereas the yellow pigments in the sample do not bind. Removal of the pigments serves a dual purpose. It is a prerequisite for binding of P450 enzyms to the subsequent columns, and it enables assessment of the content of P450 by spectrometry (carbon monoxide and substrate binding). The present invention demonstrates that P450OX in contrast shows a low binding affinity to the DEAE column and is essentially recovered in the run through and wash fractions. To separate P450OX activity from the yellow pigments by a Triton X-114 based phase partitioning procedure is applied. Using preferentially 0.6 to 1% Triton X-114, P450OX is found to partition to both phases in contrast to P450TYR, which is recovered in the detergent rich upper phase. By increasing the concentration of Triton X-114 up to 6%, the majority of P450OX is recovered from the detergent poor lower phase, while the yellow pigments are present in the upper phase. A disadvantage of using 6% Triton X-114 is an enhancement of the conversion of P450OX into its denatured P420 form. This knowledge is used in the present invention to purify for the first time P450II monooxygenases such as P450OX, to clone the genes encoding the monooxygenases, and to stably transform plants with the monooxygenase encoding genes. The isolation of P450OX and determination of partial amino acid sequences permit the design of oligonucleotide probes and the isolation of a cDNA encoding P450OX. However, in the present case cloning was accomplished via an independent approach.
The invention relates primarily to DNA molecules encoding cytochrome P450II monooxygenases, which catalyze the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrin. Preferably the aldoxime is the product of a conversion of an amino acid selected from the group consisting of tyrosine, phenylalanine, tryptophan, valine, leucine, isoleucine and cyclopentenylglycine or an amino acid selected from the group consisting of L-tyrosine, L-valine and L-isoleucine, catalyzed by a P450I monooxygenase as described in WO 95/16041. The DNA molecules according to the invention either correspond to naturally occuring genes or to homologues thereof which are the result of mutation, deletion, truncation, etc. but still encode a cytochrome P450II monooxygenase, which catalyzes the conversion of an aldoxime to a nitrile and the subsequent conversion of said nitrile to the corresponding cyanohydrin. The monooxygenases according to the invention catalyze more than one reaction of the biosynthetic pathway of cyanogenic glycosides and preferably contain a single catalytic center.
Cytochrome P450II enzymes might be present in most living organisms. The DNA molecules according to the present invention encoding P450II monooxygenases are structurally and functionally similar to DNA molecules obtainable from various plants which produce cyanogenic glycosides. In a preferred embodiment of the invention the DNA molecules hybridize to a fragment of the DNA molecule with the nucleotide sequence given in SEQ ID NO:1. Said fragment is more than 10 nucleotides long and preferably longer than 15, 20, 25, 30, or 50 nucleotides. Factors that affect the stability of hybrids determine the stringency of hybridization conditions and can be measured in dependence of the melting temperature Tm of the hybrids formed. The calculation of Tm is desribed in several textbooks. For example Keller et al describe in: xe2x80x9cDNA Probes: Background, Applications, Proceduresxe2x80x9d, Macmillan Publishers Ltd, 1993, on pages 8 to 10 the factors to be considered in the calculation of Tm values for hybridization reactions. The DNA molecules according to the present invention hybridize with a fragment of SEQ ID NO:1 at a temperatur 30xc2x0 C. below the calculated Tm of the hybrid to be formed. Preferably they hybridize at temperatures 25, 20, 15, 10, or 5xc2x0 C. below the calculated Tm.
For the purposes of gene manipulation using recombinant DNA technology the DNA molecule according to the invention may in addition to the gene coding for the monooxygenase comprise DNA which allows for example replication and selection of the inventive DNA in microorganisms such as E. coli, Bacillus, Agrobacterium, Streptomyces or yeast. It may also comprise DNA which allows the monooxygenase genes to be expressed and selected in homologous or heterologous plants. Such sequences comprise but are not limited to genes the codon usage of which has been adapted to the codon usage of the heterologous plant as described in WO93/07278; to genes conferring resistance to neomycin, kanamycin, methotrexate, hygromycin, bleomycin, streptomycin, or gentamycin, to aminoethylcystein, glyophosphate, sulfonylurea, or phosphinotricin; to scorable marker genes such as galactosidase; to its natural promoter and transcription termination signals; to promoter elements such as the 35S and 19S CaMV promoters, or tissue specific plant promoters such as promoters specific for root (described for example in EP-452269-A2, WO91/13992, U.S. Pat. No. 5,023,179), green leaves such as the maize phosphoenol pyruvate carboxylase (PEPC), pith or pollen (described for example in WO93/07278), or inducible plant promoters (EP-332104); and to heterologous transcription termination signals.
The present invention also relates to the P450II monooxygenases which catalyze the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrine. In a preferred embodiment of the invention the monooxygenases are purified and can be used to establish monoclonal or polyclonal antibodies which specifically bind to the monooxygenases. In particular cytochrome P450OX having a molecular weight of 55 kD as determined by SDS-PAGE is isolated from Sorghum bicolor (L.) Moench. Its amino acid sequence is given in SEQ ID NO:2.
The catalytic properties of P450OX resembles those of a cytochrome P450 activity reported in microsomes from rat liver (DeMaster et al, J. Org. Chem. 5074-5075, 1992). A characteristic of cytochrome P450OX and of other members belonging to the cytochrome P450OX family is that dehydration of the aldoxime to the corresponding nitrile is dependent on the presence of NADPH but that this dependence in some cases can be overcome by the addition of sodium dithionite or other reductants.
Of all known sequences for cytochrome P450 enzymes, cytochrome P450OX shows the highest amino acid sequence identity (44%) to the avocado enzyme CYP71A1 and less than 40% identity to all other members of the CYP71 family. Avocados, do not produce cyanogenic glycosides and CYP71A1 does not catalyze the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrin. Thus, according to the present invention a family of cytochrome P450II monooxygenases can be defined the members of which catalyze the conversion of an aldoxime to the corresponding cyanohydrin and have a 40% or higher amino acid sequence identity to that of cytochrome P450OX. Preferably the amino acid sequence identity with cytochrome P450OX is higher than 50% or higher than 55%. It is suggested to assign P450OX the first member of a new CYP71 subfamily (CYP71E1) as it clusters with other CYP71 sequences in dendrograms, the graphical output of a multiple sequence alignment. Generally, according to the nomenclature commitee, less than 40% sequence identity on the amino acid level is required for a cytochrome P450 to be assigned to a new CYP family and sequences that are more than 55% identical are assigned to the same subfamily. When making multiple sequence alignments not only sequence identities but also sequence similarities such as same net charge or a comparable hydrophobicity/hydrofilicity of the individual amino acids are considered. In such alignments P450OX clusters with the other CYP71 sequences and should therefore be included in the CYP71 family despite the fact that it shows less than 40% identity to all other members of the CYP71 family except CYP71A1 from avocado. As it shows low sequence identity to the other members it ought to be assigned to a new subfamily. The other CYP71 family members are all from non-cyanogenic species and their function is unknown. The catalytic properties of the previously identified P450s belonging to the CYP71 family remain elusive. They are thought to be involved in terpene hydroxylations. None of them has been suggested to utilize oximes as substrates nor to be multifunctional converting aldoximes into nitriles and cyanohydrins.
A further embodiment of the present invention is to be seen in a method for the preparation of cDNA coding for a cytochrome P450II monooxygenase, which catalyzes the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrin. It comprises
(a) isolating and solubilizing microsomes from plant tissue producing cyanogenic glycosides,
(b) purifying the cytochrome P450 monooxygenase,
(c) raising antibodies against the purified monooxygenase,
(d) probing a cDNA expression library of plant tissue producing cyanogenic glycosides with said antibody, and
(e) isolating clones which express the monooxygenase.
Microsomes can be isolated from plant tissues which show a high activity of the enzyme system responsible for biosynthesis of the cyanogenic glycosides. These tissues may be different from plant species to plant species. A preferred source of microsomes are freshly isolated shoots harvested 1 to 20 days, preferably 2 to 10 days and most preferably 2 to 4 days after germination. Etiolated seedlings are preferred from plant producing cyanogenic glycosides but light grown seedlings may also be used. Following isolation the microsomes are solubilized in buffer containing one or more detergents. Preferred detergents are RENEX 690 (J. Lorentzen A/S, Kvistgard, Denmark), reduced Triton X-100 (RTX-100), Triton X-114, and CHAPS.
The cytochrome P450 monooxygenases can be purified applying standard techniques for protein purification such as ultracentrifugation, fractionated precipitation, dialysis, SDS-PAGE and column chromatography. Possible columns comprise but are not limited to ion exchange columns such as DEAE Sepharose, Reactive dye columns such as Cibacron yellow 3 agarose, Cibacron blue agarose and Reactive red 120 agarose, and gel filtration columns such as Sephacryl S-1000. The cytochrome P450 content of the individual fractions can be determined from carbon monoxide difference spectra. A special difficulty during the isolation of P450OX which also renders quantification of P450OX difficult is its co-migration with yellow pigments during the initial purification steps instead of binding to the ion exchange column normally used for purification of P450 enzymes such as for example P450TYR. The presence of yellow pigments prevents the binding of P450OX to a number of different column materials and thus constitutes a major obstacle towards further purification. Separation of P450OX from the yellow pigments could, however, be accomplished by temperature induced Triton X-114 phase partitioning. The method was optimized with respect to P450OX recovery and removal of pigments by increasing the amount of Triton X-114. At 6%, which is six to ten fold the level used for other P450s, approximately 80% of the P450OX activity partitions to the clear lower phase. Little purification besides the removal of yellow pigments is achieved in this purification step. However, when the P450OX containing lower phase is applied to a Cibacron blue dye column, salt gradient elution produced nearly homogeneous P450OX as judged from the presence of a major Coomassie stained band with an apparent molecular mass of 55 kDa in those fractions which by reconstitution showed P450OX activity. Isolated P450OX produced a carbon monoxide spectrum with an absorption peak at 450 nm but a relatively large part of the isolated enzyme was present in the denatured P420 form. Quantitative determination of the total content and specific activity of P450OX at the different steps in the isolation procedure was hampered by the continuous conversion of P450OX into the denatured P420 form. In addition, the specific activity of P450OX is dependent on the inhibitory effects exerted by the different detergents used. The total P450 content of the fractions is thus to be considered semiquantitative.
The purified proteins can be used to elicit antibodies in for example mice, goats, sheeps, rabbits or chickens upon injection. 5 to 50 xcexcg of protein are injected several times during approximately 14 day intervals. In a preferred embodiment of the invention 10 to 20 xcexcg are injected 2 to 6 times in 14 day intervals. Injections can be done in the presence or absence of adjuvants. Immunoglobulins are purified from the antisera and spleens can be used for hybridoma fusion as described in Harlow and Lane, xe2x80x98Antibodies: A Laboratory Manualxe2x80x99, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, herein incorporated by reference. Antibodies specifically binding to a cytochrome P450II monooxygenase can also be used in plant breeding to detect plants producing altered amounts of cytochrome P450 monooxygenases and thus altered amounts of cyanogenic glycosides.
The methods for the preparation of plant tissue cDNA libraries are extensively described in Sambrook et al, Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, the essential parts of which regarding preparation of cDNA libraries are herein incorporated by reference. PolyA+ RNA is isolated from plant tissue which shows a high activity of the enzyme system responsible for biosynthesis of the cyanogenic glycosides. These tissues may be different from plant species to plant species. A preferred tissue for polyA+ RNA isolation is the tissue of freshly isolated shoots harvested 1 to 20 days, preferably 2 to 10 days and most preferably 2 to 4 days after germination. The obtained cDNA libraries can be probed with antibodies specifically binding the cytochrome P450II monooxygenase and clones expressing the monooxygenase can be isolated.
An alternative method for the preparation of cDNA coding for a cytochrome P450II monooxygenase comprises
(a) isolating and solubilizing microsomes from plant tissue producing cyanogenic glycosides,
(b) purifying the cytochrome P450II monooxygenase,
(c) obtaining a complete or partial protein sequence of the monoxygenase,
(d) designing oligonucleotides specifying DNA coding for 4 to 15 amino acids of said monooxygenase protein sequence
(e) probing a cDNA library of plant tissue producing cyanogenic glycosides with said oligonucleotides, or DNA molecules obtained from PCR amplification of cDNA using said oligonucleotides, and
(f) isolating clones which encode cytochrome P450II monooxygenase.
Amino acid sequences of internal peptides which are the result of protease digestion can be obtained by standard techniques such as Edman degradation. Oligonucleotides specifying DNA coding for partial protein sequences of the inventive monooxygenases are obtained by reverse translation of parts of the protein sequence according to the genetic code. Protein sequences encoded by DNA sequences of low degeneracy are preferred for reverse translation. Their length ranges from 4 to 15 and preferably from 5 to 10 amino acids. If necessary the codons used in the oligonucleotides can be adapted to the codon usage of the plant source (Murray et al, Nucleic Acids Research 17:477-498, 1989). The obtained oligonucleotides can be used to probe cDNA libraries as described in Sambrook et al, (Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) for clones which are able to basepair with said oligonucleotides. Alternatively, oligonucleotides can be used in a polymerase chain reaction, the methodology of which is known in the art, with plant cDNA as the template for amplification. In this case the obtained amplification products are used to probe the cDNA libraries. Clones encoding cytochrome P450II monooxygenases are isolated.
An alternative method of cloning genes is based on the construction of a gene library composed of expression vectors. In that method, analogously to the methods already described above, genomic DNA, but preferably cDNA, is first isolated from a cell or a tissue capable of expressing a P450II monooxygenase and is then spliced into a suitable expression vector. The gene libraries so produced can then be screened using suitable means, preferably antibodies. Clones which comprise the desired gene or at least part of the gene as an insert are selected.
Alternatively, cDNA molecules coding for a cytochrome P450 monooxygenase which catalyzes conversion of an aldoxime to a nitrile and conversion of said nitrile to the corresponding cyanohydrin; can be achieved by
(a) designing degenerated oligonucleotides covering 3 to 10 amino acids of conserved regions of A-type cytochromes,
(b) using the degenerated oligonucleotides to amplify one or more cytochrome specific DNA fragments using the polymerase chain reaction,
(c) screening a cDNA library with the cytochrome specific fragments to obtain full length cDNA,
(d) expressing the full length cDNA in a microbial host,
(e) identifying hosts expressing cytochrome P450 monooxygenase which catalyzes the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrin, and
(f) purifying the cloned DNA from said host.
Total DNA from a DNA library, preferably from a cDNA library, can be used as template in a PCR reaction with one or more primers representing conserved regions of A-type cytochromes (Durst et al, Drug Metabolism and Drug Interactions 12: 189-206, 1995) which are believed to be derived from a common plant cytochrome P450 ancestor. Based on a multiple sequence alignment of A-type cytochromes P450 three highly conserved regions on the amino acid level can be defined: region 1 (V/I)KEX(L/F)R, region 2 FXPERF, and region 3 PFGXGRRXCXG. Degenerate inosine (I) containing primers can be designed each covering 3 to 10 and preferably about 5 or 6 amino acids of the two regions respectively. PCR is for example performed in three consecutive rounds. Round 1 using a primer covering the consensus region FXPERF and a standard T7 primer covering the T7 promoter in the library vector amplifies cDNAs derived from mRNAs encoding A-type cytochromes P450. A second round of PCR using primers covering the two consensus regions and the amplified DNA of round 1 as template preferentially amplifies a 100 bp fragment which is then ligated into pBluescript and sequenced. Gene specific primers are designed based on the DNA sequence obtained. They are used in round 3 in combination with a primer complementary to the poly A tail (primer dT+V) and DNA of PCR round 1 as the template to amplify an approximately 500 bp DNA fragment which can be used as a gene specific probe to isolate full-length cDNAs. This PCR approach is not unique to the isolation of P450OX but is general for the isolation of A-type cytochromes P450. The A-type cytochromes P450 obtained need to be heterologously expressed to determine their function.
cDNA clones or PCR products prepared as described above or fragments thereof may be used as a hybridization probe in a process of identifying further DNA sequences encoding a protein product that exhibits P450II monooxygenase activity from a homologous or a heterologous source organism such as fungi or heterologous plants. A suitable source is tissue from plants containing cyanogenic glycosides. Said clones or PCR products may also be used as an RFLP marker to determine, for example, the location of the cytochrome P450 monooxygenase gene or a closely linked trait in the plant genome or for marker assisted breeding [EP-A 306139; WO 89/07647].
Using the methods described above it is possible to isolate various genes that code for a P450II monooxygenase. Said genes can be used in a method for producing a purified recombinant cytochrome P450II monooxygenase which catalyzes the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrin; comprising
(a) engineering the gene encoding said monooxygenase to be expressible in a host organism such as bacteria, yeast or insect cells,
(b) transforming said host organism with the engineered gene, and
(c) isolating the protein from the host organism or the culture supernatant.
In a preferred embodiment of the invention the method is used to obtain purified recombinant cytochrome P450OX, or cytochrome P450OX which has been modified by known techniques of gene technology. Preferably the modifications lead to increased expression of the recombinant protein or to altered substrate specificity.
The inventive DNA molecules can be used to obtain transgenic plants resistant to insects or acarids. Specific embodiments are listed but not limited to those in Table B of WO 95/16041 (page 45) as well as to nematodes described below. For convenience only said Table is not repeated in this specification but it is meant to be incorporated herein by referring to the disclosure of WO 95/16041. Preferably the transgenic plants are resistant to Coleoptera and Lepidoptera such as western corn root worm (Diabrotica virgifera virgifera), northern corn root worm (Diabrotica longicornis barberi), southern corn rootworm (Diabrotica undecimpunctata howardi), cotton bollworm, European corn borer, corn root webworm, pink bollworm and tobacco budworm. Nematodes are the principal animal parasites of plants causing global losses to agriculture estimated at  greater than $100 billion each year. Certain nematodes induce feeding sites involving plant cell modification and feeding at one site for several hours or considerably more. They include species of the genera Meloidogyne Globodera, Heterodera, Rotylenchulus, Tylenchulus, Naccobus, Xiphinema, Longidorus, Paralongidorus, Cryphodera, Trophotylenchulus, Hemicycliophora, Criconemella, Verutus and Heliocotylenchus. Genera considered to feed for a more restricted period at one site include Pratylenchus, Radopholus, Hirschmanniella, Trichodorus, Paratrichodorus, Ditylenchus, Aphelenchoides, Scutellonema, and Belonolaimus.
The transgenic plants comprise DNA coding for the new monooxygenases which catalyze the conversion of said aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrine. In addition the transgenic plants may comprise monooxygenase genes genetically linked to herbicide resistance genes. The transgenic plants are preferably monocotyledoneous or dicotyledoneous plants. Specific embodiments are listed in Table A of WO 95/16041 (pages 33-44). For convenience only said Table is not repeated in this specification but it is meant to be incorporated herein by referring to the disclosure of WO 95/16041. Preferably they are selected from the group consisting of maize, rice, wheat, barley, sorghum, cotton, soybeans, sunflower, grasses, oil seed rape, sugar beet, broccoli, cauliflower, cabbage, cucumber, sweet corn, daikon, benas, lettuce, melon, pepper, squash, tomato, and watermelon. The plants can be obtained by a method comprising
(a) introducing into a plant cell or plant tissue which can be regenerated to a complete plant, DNA comprising a gene expressible in that plant encoding an inventive monooxygenase, and
(b) selecting transgenic plants.
Similarly the inventive DNA molecules can be used to obtain transgenic plants expressing anti-sense or sense RNA or ribozymes targeted to the genes of the endogenous P450II monooxygenases. Expression of these molecules in transgenic plants reduces the expression of cytochrome P450II monooxygenases. Such plants show improved disease resistance or nutritive value due to reduced expression of cyanogenic glycosides. The plants can be obtained with a method comprising
(a) introducing into a plant cell or tissue which can be regenerated to a complete plant, DNA encoding sense RNA, anti sense RNA or a ribozyme, the expression of which reduces the expression of cytochrome P450II monooxygenases, and
(b) selecting transgenic plants.
A number of very efficient processes are available for introducing DNA into plant cells, which processes are based on the use of gene transfer vectors or on direct gene transfer processes.
One possible method of inserting a gene construct into a cell makes use of the infection of the plant cell with Agrobacterium tumefaciens and/or Agrobacterium rhizogenes, which has been transformed with the said gene construction. The transgenic plant cells are then cultured under suitable culture conditions known to the person skilled in the art, so that they form shoots and roots and whole plants are finally formed.
Within the scope of this invention is the so-called leaf disk transformation using Agrobacterium (Horsch et al, Science 227:1229-1231, 1985). Sterile leaf disks from a suitable target plant are incubated with Agrobacterium cells comprising one of the chimaeric gene constructions according to the invention, and are then transferred into or onto a suitable nutrient medium. Especially suitable, and therefore preferred within the scope of this invention, are LS media that have been solidified by the addition of agar and enriched with one or more of the plant growth regulators customarily used, especially those selected from the group of the auxins consisting of xcex1-naphthylacetic acid, picloram, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, indole-3-butyric acid, indole-3-lactic acid, indole-3-succinic acid, indole-3-acetic acid and p-chlorophenoxyacetic acid, and from the group of the cytokinins consisting of kinetin, 6-benzyladenine, 2-isopentenyladenine and zeatin. The preferred concentration of auxins and cytokinins is in the range of 0.1 mg/l to 10 mg/l.
After incubation for several days, but preferably after incubation for 2 to 3 days at a temperature of 20xc2x0 C. to 40xc2x0 C., preferably from 23xc2x0 C. to 35xc2x0 C. and more preferably at 25xc2x0 C. and in diffuse light, the leaf disks are transferred to a suitable medium for the purpose of shoot induction. Especially preferred for the selection of the transformants is an LS medium that does not contain auxin but contains cytokinin instead, and to which a selective substance has been added. The cultures are kept in the light and are transferred to fresh medium at suitable intervals, but preferably at intervals of one week. Developing green shoots are cut out and cultured further in a medium that induces the shoots to form roots. Especially preferred within the scope of this invention is an LS medium that does not contain auxin or cytokinin but to which a selective substance has been added for the selection of the transformants.
In addition to Agrobacterium-mediated transformation, within the scope of this invention it is possible to use direct transformation methods for the insertion of the gene constructions according to the invention into plant material.
For example, the genetic material contained in a vector can be inserted directly into a plant cell, for example using purely physical procedures, for example by microinjection using finely drawn micropipettes (Neuhaus et al, Theoretical and Applied Genetics 74:363-373, 1987), electroporation (D""Halluin et al, The Plant Cell 4:1495-1505, 1992; WO92/09696), or preferably by bombarding the cells with microprojectiles that are coated with the transforming DNA (xe2x80x9cMicroprojectile Bombardmentxe2x80x9d; Wang et al, Plant Molecular Biology 11:433-439, 1988; Gordon-Kamm et al, The Plant Cell 2:603-618, 1990; McCabe et al, Bio/Technology 11:596-598, 1993; Christou et, Plant Physiol. 87:671-674, 1988; Koziel et al, Biotechnology 11: 194-200, 1993). Moreover, the plant material to be transformed can optionally be pretreated with an osmotically active substance such as sucrose, sorbitol, polyethylene glycol, glucose or mannitol.
Other possible methods for the direct transfer of genetic material into a plant cell comprise the treatment of protoplasts using procedures that modify the plasma membrane, for example polyethylene glycol treatment, heat shock treatment or electroporation, or a combination of those procedures (Shillito et al, Biotechnology 3:1099-1103,1985).
A further method for the direct introduction of genetic material into plant cells, which is based on purely chemical procedures and which enables the transformation to be carried out very efficiently and rapidly, is described in Negrutiu et al, Plant Molecular Biology 8:363-373, 1987.
Also suitable for the transformation of plant material is direct gene transfer using co-transformation (Schocher et al, Bio/Technology 4:1093-1096,1986).
The list of possible transformation methods given above by way of example does not claim to be complete and is not intended to limit the subject of the invention in any way.
The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well as to competition by weed plants, measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such a tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.
Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained which, due to their optimized genetic xe2x80x9cequipmentxe2x80x9d, yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.
In seeds production germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof. Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD(copyright)), methalaxyl (Apron(copyright)), and pirimiphosmethyl (Actellic(copyright)). If desired these compounds are formulated together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.
It is a further aspect of the present invention to provide new agricultural methods such as the methods examplified above which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.
The following examples further describe the materials and methods used in carrying out the invention and the subsequent results. They are offered by way of illustration, and their recitation should not be considered as a limitation of the claimed invention.